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Blood. 2000 Nov 15;96(10):3399-405.
In vivo kinetics of murine hemopoietic stem cells.

Abkowitz JL, Golinelli D, Harrison DE, Guttorp P.

Division of Hematology, Department of Medicine, University of Washington, Seattle, WA, USA.

We used stochastic modeling and computer simulation to study the replication, apoptosis, and differentiation of murine hemopoietic stem cells (HSCs) in vivo. This approach allows description of the behavior of an unobserved population (ie, HSCs) on the basis of the behavior of observed progeny cells (ie, granulocytes and lymphocytes). The results of previous limiting-dilution, competitive-repopulation studies in 44 mice were compared with the results of simulated transplantation studies to identify parameters that led to comparable outcomes. Using this approach, we estimated that murine HSCs replicate (on average) once every 2.5 weeks and that the frequency of murine HSCs is 8 per 10(5) nucleated marrow cells. If it is assumed that short-term repopulating cells are distinct from HSCs, that they contribute to hemopoiesis early after transplantation, and that they are independently regulated, a frequency of 4 HSCs per 10(5) nucleated marrow cells also allows simulations that best approximate the observed data. When stochastic modeling and computer simulation were applied to limiting-dilution, autologous-transplantation studies in cats heterozygous for glucose-6-phosphate-dehydrogenase, different estimates of HSC replication rate (1 per 8.3-10 weeks) and frequency (6 per 10(7) cells) were derived. Therefore, it appears that these parameters vary inversely with increased longevity, size, or both. An implication of these data is that human HSCs may be less frequent and replicate more slowly. These findings on cell kinetics have several implications.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11071634&dopt=Abstract

Blood. 2000 Nov 15;96(10):3406-13.
Stem cell factor induces phosphatidylinositol 3'-kinase-dependent Lyn/Tec/Dok-1 complex formation in hematopoietic cells.

van Dijk TB, van Den Akker E, Amelsvoort MP, Mano H, Lowenberg B, von Lindern M.

Institute of Hematology, Erasmus University Rotterdam, Rotterdam, The Netherlands. vandijema.fgg.eur.nl

Stem cell factor (SCF) has an important role in the proliferation, differentiation, survival, and migration of hematopoietic cells. SCF exerts its effects by binding to cKit, a receptor with intrinsic tyrosine kinase activity. Activation of phosphatidylinositol 3'-kinase (PI3-K) by cKit was previously shown to contribute to many SCF-induced cellular responses. Therefore, PI3-K-dependent signaling pathways activated by SCF were investigated. The PI3-K-dependent activation and phosphorylation of the tyrosine kinase Tec and the adapter molecule p62Dok-1 are reported. The study shows that Tec and Dok-1 form a stable complex with Lyn and 2 unidentified phosphoproteins of 56 and 140 kd. Both the Tec homology and the SH2 domain of Tec were identified as being required for the interaction with Dok-1, whereas 2 domains in Dok-1 appeared to mediate the association with Tec. In addition, Tec and Lyn were shown to phosphorylate Dok-1, whereas phosphorylated Dok-1 was demonstrated to bind to the SH2 domains of several signaling molecules activated by SCF, including Abl, CrkL, SHIP, and PLCgamma-1, but not those of Vav and Shc. These findings suggest that p62Dok-1 may function as an important scaffold molecule in cKit-mediated signaling.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11071635&dopt=Abstract

Blood. 2000 Nov 15;96(10):3414-21.
An in vivo competitive repopulation assay for various sources of human hematopoietic stem cells.

Rosler ES, Brandt JE, Chute J, Hoffman R.

Hematology-Oncology Section, University of Illinois College of Medicine, Chicago, IL, USA.

The marrow repopulating potential (MRP) of different sources of human hematopoietic stem cells (HSCs) was directly compared using an in vivo assay in which severe combined immunodeficient disease (SCID) mice were implanted with human fetal bones. HSCs from 2 human lymphocyte antigen (HLA)-mismatched donors were injected individually or simultaneously into the fetal bones of a 3rd distinct HLA type and donor and recipient myeloid and lymphoid cells were identified after 8 to 10 weeks. The study compared the MRP of umbilical cord blood (CB) and adult bone marrow (ABM) CD34(+) cells as well as grafts of each type expanded ex vivo. Equal numbers of CB and ABM CD34(+) cells injected individually demonstrated similar abilities to establish multilineage hematopoiesis. However, when CB and ABM cells were transplanted simultaneously, the engraftment of CB cells was markedly superior to ABM. CB and ABM CD34(+) cells were expanded ex vivo using either a porcine microvascular endothelial cell (PMVEC)-based coculture system or a stroma-free expansion system. Primary CB CD34(+) cells or CD34(+) cells expanded in either culture system demonstrated a similar ability to engraft. However, the MRP of expanded grafts simultaneously injected with primary CB cells was uniformly inferior to primary CB cells. CD34(+) cell grafts expanded in the stroma-free system, furthermore, outcompeted CD34(+) cells expanded using the PMVEC coculture system. The triple HLA-mismatched SCID-hu model represents a novel in vivo stem cell assay system that permits the direct demonstration of the functional consequences of ex vivo HSC expansion and ontogeny-related differences in HSCs.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11071636&dopt=Abstract

Blood. 2000 Nov 15;96(10):3422-30.
SCF and G-CSF lead to the synergistic induction of proliferation and gene expression through complementary signaling pathways.

Duarte RF, Frank DA.

Department of Adult Oncology, Dana-Farber Cancer Institute, Brigham and Women's Hospital, Boston, MA, USA.

Stem cell factor (SCF) is a potent costimulatory molecule for many cytokines. Its synergy with granulocyte colony-stimulating factor (G-CSF) results in important biologic and clinical effects, although the mechanism by which this occurs remains poorly understood. To investigate this interaction, this study used a retroviral vector to transduce the G-CSF receptor into MO7e cells, which are known to express the SCF receptor. The transduced G-CSF receptor is functionally active, and the resultant MO7e-G cells recapitulate the proliferative synergy between SCF and G-CSF. When treated with both cytokines, a marked shortening of the G(0)/G(1) phase of the cell cycle occurs, associated with a suppression of the cyclin-dependent kinase inhibitor p27(kip-1). In addition, SCF and G-CSF induce the synergistic activation of c-fos, a proto-oncogene involved in propagation of mitogenic signals in hematopoietic cells. G-CSF, but not SCF, induces the tyrosine phosphorylation of STAT1 and STAT3, transcription factors that can mediate the induction of c-fos. However, SCF induces phosphorylation of STAT3 on serine727 (ser727), which is necessary for maximal STAT transcriptional activity, and the combination of SCF and G-CSF leads to complete STAT3 phosphorylation on ser727. The pathways by which SCF and G-CSF lead to serine phosphorylation of STAT3 are distinct and are partially dependent on phosphatidylinositol-3 kinase and ERKs, pathways that are also necessary for the synergistic effects of SCF and G-CSF on proliferation and c-fos induction. Thus, MO7e-G cells provide a powerful system in which the molecular basis of the synergy between SCF and G-CSF can be dissected.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11071637&dopt=Abstract

Blood. 2000 Nov 15;96(10):3522-8.
Immunophenotypic analysis of B cells in PNH: insights into the generation of circulating naive and memory B cells.

Richards SJ, Morgan GJ, Hillmen P.

Haematological Malignancy Diagnostic Service, Leeds General Infirmary, Leeds, United Kingdom. stephenathology.leeds.ac.uk

Peripheral blood B cells in patients with paroxysmal nocturnal hemoglobinuria (PNH) comprise variable mixtures of normal B cells produced before the onset of disease and glycosylphosphatidylinositol (GPI)-deficient B cells derived from the PNH hematopoietic stem cell. In a detailed phenotypic analysis of 29 patients with PNH, this study shows consistent phenotypic differences between PNH B cells and residual normal B cells. In the majority of patients with active disease, PNH B cells comprised mainly naive cells with a CD27(-)IgM(+)IgD(strong+)IgG(-) phenotype. The proportion of CD27(+) memory cells within this compartment was related to disease duration (Spearman [r(s)] 0.403; P =.030). In PNH patients with predominantly GPI-deficient hematopoiesis, that is, a large granulocyte PNH clone, the residual normal B cells had a predominantly memory (CD27(+)) phenotype. Furthermore, the majority of these memory B cells were not immunoglobulin (Ig) class switched and had an IgM(+)IgD(+)IgG(-) phenotype. Using PNH as a novel model with which to study B lymphopoiesis, this study provides direct evidence that production of new naive B cells occurs throughout life and that the major population of long-lived memory B cells are IgM(+)IgD(+). Moreover, studies of GPI(-) B cells in 2 patients in remission from PNH suggest that the life span of a B-cell clone can be more than 24 years.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11071650&dopt=Abstract

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